In blood vessels, vascular smooth muscle cells and pericytes, which are generically referred to as vascular mural cells, adhere to vascular endothelial cells from the outer surface of the vascular endothelial cells either directly or via the extracellular matrix. The ratios of adhesion of endothelial cells and parietal cells differ depending on the size of the vessel diameter, and in large vessels, multiple of layers of mural cells line a single layer of endothelial cells, in medium and small vessels, a single parietal cell lines a single endothelial cell, while in narrow-diameter vessels, a single parietal cell adheres to a plurality of endothelial cells. In this manner, lining of vascular endothelial cells by parietal cells is important for the structural “maturation” process of blood vessels. In addition, the formation of focal adhesion between endothelial cells by adhesion between vascular endothelial cells so that intravascular environmental factors (cellular and humoral factors) do not easily leak outside blood vessels is important for the functional “maturation” process of blood vessels. Moreover, blood vessels regulate blood flow so that oxygen and nutrients adequately reach all tissues by increasing blood flow through dilation of the lumen in response to demands for oxygen and nutrients by tissues, and particularly when there is a shortage thereof. In other words, the process by which the vessel lumen is controlled to be large, medium or small through structural stabilization by controlling permeability as a result of vascular endothelial cells forming focal adhesion and inducing structural stabilization due to the accompanying lining of parietal cells with vascular endothelial cells is defined as “vascular maturation”.
In addition, vessels having impaired vessel structures are formed in various disease states, and immature vessels are formed in which adhesion between endothelial cells is inhibited or adhesion of parietal cells to endothelial cells is deficient. This leads to unregulated increases in vascular permeability, resulting in abnormalities in exchanges between humoral factors and cells in tissue and blood vessels. Increased permeability results in the formation of tissue edema, which in turn causes impaired tissue function and leads to inflammation due to unregulated infiltration of inflammatory cells. Moreover, since parietal cells inhibit vascular sprouting from existing vessels, sprouting of vessels from vessels not having parietal cells becomes excessive and unregulated vessel sprouting is induced that results in exacerbation of the disease state. This type of phenomenon is observed in diseases represented by diabetic retinopathy, tumors and inflammations. In other words, the returning of blood vessels in which vascular permeability has been impaired or abnormal blood vessels that cause unregulated vascular hyperplasia to a normal state by enhancing adhesion between endothelial cells and promoting lining of endothelial cells with parietal cells is defined as “vascular normalization”. In addition, in abnormal blood vessels as described above, changes in circulatory internal and external environmental factors caused by diabetes, hyperlipemia or hypertension and the like impart damage (such as cell death) to endothelial cells and parietal cells, and are triggered by excessive increases in production of angiogenesis promoting factors caused by cancer or inflammation. When such a disease state has occurred, inhibiting damage to existing vessels and inhibiting dissociation between endothelial cells or inhibiting dissociation between endothelial cells and parietal cells is defined as “vascular stabilization”. In addition, a mechanism that inhibits cell death of endothelial cells is included in this stabilization.
Angiogenesis refers to a phenomenon by which a network of new blood vessels is formed from existing vessels, and is intimately involved with diseases primarily associated with vascular lesions, such as tumors, chronic rheumatoid arthritis, diabetic retinopathy, hyperlipemia or hypertension. Following the molecular cloning of vascular endothelial growth factor (VEGF), molecules of the VEGF family and angiopoietin (Ang) family have successively been identified as factors that specifically act on blood vessel formation. VEGF and its receptors are involved in an extremely wide range of vessel formation, extending from the initial formation of blood vessels referred to as vasculogenesis to its subsequent angiogenesis. On the other hand, Ang functions in lumen formation accompanying cellular phenomena such as sprouting, branching, intussusception or pruning by vascular endothelial cells following vasculogenesis. Ang controls adhesion between vascular endothelial cells and vascular parietal cells in the manner of peripheral cells (pericytes) and vascular smooth muscle cells through a receptor-type tyrosine kinase Tie (tyrosine kinase with Ig and EGF homology domain)-2 that is expressed in vascular endothelial cells, and functions in structural stabilization of blood vessels (Experimental Medicine, Vol. 20, No. 8 (2002), pp. 52-57: Non-Patent Document 1).
Four angiopoietin isoforms, consisting of Ang-1 to Ang-4, have previously been known, and although Ang-1 and Ang-2 are present in both humans and mice, Ang-3 is only present in mice while Ang-4 is only present in humans. Ang-1 and Ang-4 secreted from parietal cells induce adhesion between endothelial cells and parietal cells accompanying stimulation of Tie-2, induction of autophosphorylation of the intracellular tyrosine kinase domain, activation of integrin, activation of focal adhesion kinase (FAK) and activation of phosphatidylinositol-3-kinase/serine-threonine kinase (PI3K/Akt). In a normal oxygen state, although adhesion between endothelial cells and parietal cells is maintained by Ang-1 and Ang-4 constantly secreted by parietal cells, when a local hypoxic state occurs, production of Ang-1 and Ang-4 antagonists in the form of Ang-2 and Ang-3 increases, thereby temporarily inhibiting activation of Tie-2 and inhibiting adhesion between endothelial cells and the parietal cells that line them. Endothelial cells then proliferate and begin sprouting angiogenesis due to dissociation of parietal cells, which leads to the formation of a new vascular network. Since Tie-2 activation induces adhesion between endothelial cells and parietal cells, it contributes to stabilization of vessel structure, while also controlling vascular permeability by promoting adhesion between endothelial cells. In addition, since activation of Tie-2 is also known to inhibit cell death of endothelial cells (Cho, C. H., Kammerer, R. A., Lee, H. J., Yasunaga, K., Kim, K. T., Choi, H. H., Kim, W., Kim, S. H., Park, S. K., Lee, G. M. and Koh, G. Y.: Designed angiopoietin-1 variant, COMP-Ang1, protects against radiation-induced endothelial cell apoptosis, Proc. Natl. Acad. Sci. U.S.A., 2004, Apr. 13, 101(15), 5553-8: Non-Patent Document 2), blood vessels can be stabilized and normalized in response to environmental factors that damage various intracellular and extracellular vascular structures by inducing activation of Tie-2 and inhibiting blood vessel instability. In addition, by inducing activation of Tie-2 in blood vessels that have been formed by vascular endothelial cells in vascular regenerative therapy, blood vessel maturation is possible by inducing adhesion between endothelial cells and parietal cells. In addition, in diseases such as tumors or diabetic retinopathy, in which blood vessels exhibit unregulated growth as a result of parietal cells not adhering to endothelial cells, activation of Tie-2 makes it possible to normalize blood vessels by causing parietal cells to adhere to endothelial cells. In addition, according to the literature (Thurston, G., Suri, C., Smith, K., McClain, J., Sato, T. N., Yancopoulos, G. D. and McDonald, D. M.: Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1, Science 1999, Dec. 24, 286 (5449): Non-Patent Document 3), activation of Tie-2 has been reported to dilate vascular lumen, and in ischemic diseases occurring caused by vascular contraction or inhibition of vascular dilation, disease state can be improved through dilation of vascular lumen by activation of Tie-2.
In addition, a plurality of molecules having a coiled-coiled domain and fibrinogen-like domain, which are structural characteristics of Ang, have recently been discovered. Since these do not have the ability to bind to Tie-1 receptors or Tie-2 receptors, they are considered to constitute a group of molecules that differs from the existing Ang family, have been named angiopoietin-like protein (Angpt1), and Angpt-1, -2, -3, -4, -5, -6 and -7 have been reported. Although these Angpt1 are orphan ligands for which receptors have yet to be identified, they are expected to demonstrate various actions.
Activation of Tie-2 is also known to induce a dormant state in cells other than vascular endothelial cells. According to previous reports, activation of Tie-2 in hematopoietic stem cells has been reported to induce dormancy in the hematopoietic stem cells (Arai, F., Hirao, A., Ohmura, M., Sato, H., Matsuoka, S., Takubo, K., Ito, K., Koh, G. Y. and Suda, T.: Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche, Cell, 2004, Jul. 23, 118(2), 149-161: Non-Patent Document 4). In other words, induction of Tie-2 makes it possible to maintain survival of hematopoietic stem cells in vitro for long periods of time. In addition, according to previous reports, activation of Tie-2 is known to induce adhesion of cells to the extracellular matrix and the like by activating adhesion factors such as integrin (Takakura, N., Huang, X. L., Naruse, T., Hamaguchi, I., Dumont, D. J., Yancopoulos, G. D. and Suda, T.: Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis, Immunity, 1998, Nov., 9(5), 677-86: Non-Patent Document 5). Induction of this cell adhesion is thought to enable induction of maintenance of anchorage-dependent survival in hematopoietic stem cells both in vitro and in vivo due to activation of Tie-2. Moreover, according to recent reports, expression of Tie-2 has been suggested in cancer stem cells, considered to have the highest degree of malignancy in cancer tissue and be involved in cancer relapse (Lee, O. H., Xu, J., Fueyo, J., Fuller, G. N., Aldape, K. D., Alonso, M. M., Piao, Y., Liu, T. J., Lang, F. F., Bekele, B. N. and Gomez-Manzano, C.: Expression of the receptor tyrosine kinase Tie2 in neoplastic glial cells is associated with integrin beta1-dependent adhesion to the extracellular matrix, Mol. Cancer Res., 2006, Dec., 4(12), 915-26: Non-Patent Document 6). In the same manner as activation of Tie-2 is able to cause the cell cycle of hematopoietic stem cells to enter a dormant state, proliferation of cancer stem cells can also be inhibited by activation of Tie-2 expressed in cancer stem cells.